This study uses stable isotope techniques to evaluate iron metabolism in premature infants given Fe supplements in order to identify those infants who might benefit from an early Fe supplementation by demonstrating high levels of Fe absorption and RBC incorporation. The rate of absorption and red blood cell (RBC) incorporation of Fe by infants fed an iron supplemented formula designed for premature infants is unknown. We measured RBC Fe incorporation and fractional Fe absorption in 8 premature infants (birth weight 1.1 +/- 0.2 kg, gestational age 29 +/- 1 wk, weight at start of study 1.6 +/- 0.2 kg) using a novel triple isotope technique in which 57Fe was given mixed with 2 feedings of Enfamil Premature Formula (EPF) (Mead Johnson, Evansville, IN), 54Fe was given as a supplement between feeds with a multivitamin preparation, and 2 wk later 58Fe was given intravenously (IV). RBC incorporation was calculated from the enrichment of each isotope in a blood sample collected 14-15 days after isotope administration using a blood volume estimate of 80 ml/kg. Fractional absorption was calculated by dividing the RBC incorporation of the 54Fe and 57Fe by the RBC incorporation of the 58Fe. All samples were analyzed for isotope ratios using magnetic sector thermal ionization mass spectrometry. Differences between RBC incorporation and percent Fe absorption from EPF vs the supplement were marginally statistically significant (P=0.06 and P=0.07, respectively). The absorption of 54Fe and 57Fe were correlated (r=0.75, P=0.03). 57Fe RBC incorporation was negatively correlated with serum ferritin (r=-0.59, P=0.12) and positively correlated with the reticulocyte count (r=0.63, P=0.09). Relatively high rates of RBC incorporation of the IV dose may relate to the large size of the infants in this study and minimal transfusions they had received. These preliminary results indicate that Fe absorption from Fe-fortified high mineral-containing perterm formula is not markedly different from that of Fe given separately. Supported in part by Bristol-Myers Squibb Company.